Dose dependent translocations of fluorescent probes of PIP₂hydrolysis in cells exposed to nanosecond pulsed electric fields

Abstract

Previously, it was demonstrated that small nanometer-sized pores (nanopores) are preferentially formed after exposure to nanosecond pulsed electric fields (nsPEF). We have reported that nanoporation of the plasma membrane directly affects the phospholipids of the cell membrane, ultimately culminating in phosphatidylinositol 4,5 -bisphosphate (PIP 2 ) intracellular signaling. PIP 2 , located within the internal layer of the plasma membrane, plays a critical role as a regulator of ion transport proteins, a source of second messenger compounds, and an anchor for cytoskeletal elements. In this proceeding, we present data that demonstrates that nsPEFs initiate electric field dosedependent PIP 2 hydrolysis and/or depletion from the plasma membrane through the observation of the accumulation of inositol 1,4,5 -trisphosphate (IP 3 ) in the cytoplasm and the increase of diacylglycerol (DAG) on the inner surface of the plasma membrane. The phosphoinositide signaling cascade presented here involves activation of phospholipase C (PLC) and protein kinase C (PKC), which are responsible for a multitude of biological effects after nsPEF exposure. These results expand our current knowledge of nsPEF induced physiological effects, and serve as a basis for development of novel tools for drug independent stimulation or modulation of different cellular functions.

Characterization of Pressure Transients Generated by Nanosecond Electrical Pulse (nsEP) Exposure

“…These single intense shear stress waves, termed “μtsunami”, were also reported to directly or indirectly stimulate specific G-protein coupled receptors (GPCR) on the plasma membrane leading to the production of IP 3 36 . Tolstykh et al has shown that nsEP exposure activates the intracellular phosphoinositide signaling pathway 37 38 39 , hypothetically through the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) or PIP 2 , a well-characterized intracellular pathway that originates on the inner surface of the plasma membrane. Hydrolysis of PIP 2 ultimately causes intracellular calcium release from the endoplasmic reticulum via inositol trisphosphate (IP 3 ) receptors, activating protein kinase C (PKC).…”